Recombinant DNA technology and genetic engineering have made it possible to introduce desired DNA sequences into plant cells to allow for the expression of proteins of interest. The relative ease of obtaining commercially viable transformation events in important crops, however, remains a challenge.
Transformation of plant cells and tissues with foreign DNA can be achieved in a number of ways known to the art. For example, (a) particle bombardment of cultured cells (Gordon-Kamm et al., 1990, and U.S. Pat. No. 5,886,244), immature embryos (Koziel et al., 1993), meristems (Lowe et al., 1995); (b) electroporation of immature embryos (D'Halluin et al., 1992), cultured cells (Laursen et al., 1994); (c) electroporation and/or polyethylene glycol treatment of protoplasts (Rhodes et al., 1989; Omirulleh et al., 1993), and (d) co-cultivation with Agrobacterium tumefaciens (Ishida et al., 1996; Hiei et. al., 1997; Zhao et al., 1998). See also U.S. Pat. No. 6,706,394, which relates to the use of magnetizable microparticles and magnetic fields for transformation.
Cho et al. (Plant Science 138 [1998] 229-244) relates to a system for transformation of barley. Cho et al. (Plant Science 148 [1999] 9-17) relates to a system for transformation of oats. Cho et al. (Plant Cell Reports [2000] 19:1084-1089) relates to the production of transgenic fescue by particle bombardment. Cho et al. (Plant Cell Reports [2001] 20: 318-324) relates to transformed orchardgrass. U.S. 20010031496 A1 and U.S. Pat. No. 6,235,529 relate to plant transformation and regeneration. U.S. Pat. Nos. 5,736,369 and 6,486,384 relate to the transformation of cereals. U.S. Pat. No. 6,140,555 relates to maize transformation. Zhang et al. (Plant Cell Reports [1999] 18: 959-966) relates to an oat transformation system that uses high concentrations of mannitol and sorbitol (0.2M) as osmotic treatments prior to particle bombardment. This is done to partially desiccate the cells so that they do not burst upon impact by the particles.
Plant cells can be grown in isolation from intact plants in tissue culture systems. Plant tissue cultures can be initiated from almost any part of a plant. Pieces of plant tissue will slowly divide and grow into a colorless mass of cells if they are kept in special in vitro culture conditions. The cells have the characteristics of callus cells, rather than other plant cell types. Callus cells appear on cut surfaces when a plant is wounded; these cells gradually cover and seal the damaged area.
Tissue culture cells generally lack the distinctive features of most plant cells. They have a small vacuole, and lack chloroplasts and photosynthetic pathways; structural or chemical features that distinguish many cell types within the intact plant are absent. They are most similar to the undifferentiated cells found in meristematic regions; the cells become fated to develop into each cell type as the plant grows. Tissue cultured cells can also be induced to re-differentiate into whole plants by alterations to the growth media.
Totipotency is the ability of undifferentiated plant cells to develop, in vitro, into whole plants or plant organs, when given the optimum in vitro culture conditions. Totipotent cells that undergo rapid division are generally regarded as highly suitable targets for introduction of DNA as a first step in the generation of transgenic plants. In corn, one prolific source of such cells is the so-called Type II callus (Armstrong and Green, 1985).
In maize, totipotent cell cultures typically proliferate in vitro as clusters of non-green cells and only synthesize chlorophyll in mature chloroplasts upon shoot differentiation during plant regeneration. However, green or chlorophyllous cultures organize plastome structures in the presence of light and develop chloroplasts. The cell cultures of photoautotrophic or photomixotrophic cells have functional chloroplast in a sugar-free or minimal medium, respectively. Such cultures are common in dicots, and several plants such as soybean, tobacco, Chenopodium, Datura, and cotton can be used for making such cultures routinely. However, such cultures are rare or nonexistent for most or all monocots, with an exception of blue grama grass (green embryogenic suspension cells) (Aguado-Santacruz et al. 2001: Plant Cell Rep 20: 131-136). There are few, if any, other reports of monocots where some green callus/tissue development was achieved. Any such attempts were typically aimed at improving regeneration or improved recovery of transgenic plants.
The plastids of higher plants are an attractive target for genetic engineering. Chloroplast (a type of plastid) transformation has been achieved and is advantageous. See e.g. U.S. Pat. Nos. 5,932,479; 6,004,782; and 6,642,053. See also U.S. Pat. Nos. 5,693,507 and 6,680,426. Advantages of transformation of the chloroplast genome include:                1) potential environmental safety because transformed chloroplasts are only maternally inherited and thus are not transmitted by pollen out crossing to other plants;        2) the possibility of achieving high copy number of foreign genes; and        3) eduction in plant energy costs because importation of proteins into chloroplasts, which is highly energy dependent, is reduced.        
Plant plastids (chloroplasts, amyloplasts, elaioplasts, etioplasts, chromoplasts, etc.) are the major biosynthetic centers that, in addition to photosynthesis, are responsible for producing many industrially important compounds such as amino acids, complex carbohydrates, fatty acids, and pigments. Plastids are derived from a common precursor known as a proplastid; thus, the plastids in a given plant species all have the same genetic content.
Plastids of most plants are maternally inherited. Consequently, unlike heterologous genes expressed in the nucleus, heterologous genes expressed in plastids are not disseminated in pollen. Therefore, a trait introduced into a plant plastid will not be transmitted to wild-type relatives. This offers an advantage for genetic engineering of plants for tolerance or resistance to natural or chemical conditions, such as herbicide tolerance, as these traits will not be transmitted to wild-type relatives.
The plastid genome (plastome) of higher plants is a circular double-stranded DNA molecule of 120-160 kb which may be present in 1,900-50,000 copies per leaf cell (Palmer, 1991). In general, plant cells contain 500-10,000 copies of a small 120-160 kilobase circular genome, each molecule of which has a large (approximately 25 kb) inverted repeat. Thus, it is possible to engineer plant cells to contain up to 20,000 copies of a particular gene of interest; this can potentially result in very high levels of foreign gene expression.
Stable transformation of the tobacco plastome has been achieved through the following steps: (i) introduction of transforming DNA, encoding antibiotic resistance, by the biolistic process (Svab et al. 1990; Svab and Maliga 1993) or PEG treatment (O'Neill et al., 1993), (ii) integration of the transforming DNA by two homologous recombination events and (iii) selective elimination of the wild-type genome copies during repeated cell divisions on a selective medium. Spectinomycin resistance has been used as a selective marker encoded either in mutant plastid 16S ribosomal RNA genes (Svab et al. 1990; Staub and Maliga 1992), or conferred by the expression of an engineered bacterial aadA gene (Svab and Maliga 1993). Vectors that utilize aminoglycoside adenyltransferase (aadA) as a selectable marker gene, and target the insertion of chimeric genes into the repeated region of tobacco plastome, are available (Zoubenko et al., 1994). Selection of plastid transformants by kanamycin resistance, based on the expression of neomycin phosphotransferase, is more difficult but also feasible (Carrer et al., 1993).
Until recently, successful plastid transformation techniques for higher plants have been limited to model crop plants such as tobacco (U.S. Pat. No. 5,451,513; Svab et al. (1990), Proc. Natl. Acad. Sci. USA 87: 8526-8530 and Svab et al. (1993), Proc. Natl. Acad. Sci. USA 90: 913-197) and Arabidopsis (Sikdar, et al. (1998) Plant Cell Reports 18: 20-24). A review of plastid transformation of flowering plants is provided by Maliga (1993) Trends in Biotech. 11: 101-107.
Furthermore, the methods described for Arabidopsis plants, produce infertile regenerates. PCT Publication WO 97/32977 also describes methods for the plastid transformation of Arabidopsis and provides prophetic examples of plastid transformation of Brassica plastids. However, transplastomic Brassica plants were not produced using the methods described therein. U.S. Pat. No. 6,515,206 relates to plastid transformation of Brassica. 
Plastomic transformation was extended to potatoes and tomatoes (see e.g. Sidorov et al., The Plant Journal, vol. 19, iss. 2, page 209 (July 1999); and Ruf et al., Nature Biotechnology, vol. 9, no. 9, pp. 870-875 (September 2001); respectively). However, these plants are closely related to tobacco, and it is possible to work the tobacco protocol to produce transplastomic tomato and potato. Thus, one would not have expected to apply the tobacco technology beyond the tobacco family (Nicotinaceae) to crop plants.
More recently, three more plants have been shown to be susceptible to plastomic transformation: cotton, carrots, and soybeans (see e.g. Kumar and Daniell, Plant Molecular Biology, “Manipulation of gene expression facilitates cotton plastid transformation of cotton by somatic embryogenesis and maternal inheritance of transgenes” in press (2004); Kumar and Daniell, Plant Physiology, “Plastid expressed betaine aldehyde dehydrogenase gene in carrot cultured cells, roots and leaves confers enhanced salt tolerance,” in press (2004); and Nathalie Dufourmantel, Bernard Pelissier, Frederic Garcon, Gilles Peltier, Jean-Marc Ferullo, Ghislaine Tissot, “Generation of fertile transplastomic soybean,” Plant Molecular Biology, Volume 55, Issue 4, July 2004, Pages 479-489). The use of tissues other than leaf explants as target material has been demonstrated in all three of these plants, where the target material was embryos or embryogenic callus. However, there appears to be limitations in the selectable markers that could be successfully used to achieve plastomic transformation. This is currently limited to only Spectinomycin, aphA6/npt (neomycin class antibiotic), and EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) marker (including the other ones glyphosate oxido -reductase (GOX) and the aroA gene see U.S. Pat. No. 4,535,060). Biolistics is the most preferred DNA delivery method that is used to enable this technology in these system. PEG mediated delivery has been also reported, but not widely used to transform plastids.
With possibly one exception of blue grama grass (Aguado-Santacruz et al. 2001: Plant Cell Rep 20: 131-136), no other monocots (including any cereal cultures) are reported to be chlorophyllous, photoautotropic, and/or photomixotropic. The methods of Aguado-Santacruz et al. were unsuccessfully applied to maize. See Example 10, below.
For corn and cereals, as well as dicots, a chlorophyllous photoautotropic suspension system that remains embryogenic and could regenerate into plants would be ideal for photosynthetic, herbicidal, and plastid genetic manipulation studies. However, such systems have not heretofore been known in the art.